Cancer is a highly heterogeneous disease, and the development of successful treatment strategies requires an understanding of the cellular mechanisms resulting in diversity of cancer cell dynamics and interactions with the tissue environment. As a result of the ongoing and rapid development of fluorescent probes, biosensors and gene targeting approaches, in combination with remarkable advances in optical microscopy technology, quantitative microscopy has become an increasingly important tool in cancer research. Proper training in the principles of quantitative microscopy and image analysis is essential to generating reliable data, correctly interpreting experiments and enhancing the reproducibility of cancer research. The Cold Spring Harbor Laboratory proposes to continue the course Quantitative Imaging: From Cells to Molecules, to be held in the spring of 2016-2020. This two-week intensive course provides students with a thorough foundation in modern quantitative optical microscopy and image analysis. Students (ranging from graduate students to Principal Investigators) gain hands-on experience in a wide range of quantitative microscopy techniques using biological specimens, primarily cancer cell lines and model systems. A unique aspect of this microscopy course is its strong emphasis on generating accurate and precise quantitative data. Each day, students work through a focused quantitative exercise, from image acquisition through data analysis, designed to answer a specific biological question. The exercises are designed such that, in the duration of the course, the most common types of quantitative measurements used in cancer research are covered, including detection/tracking/counting of diffraction-limited objects, subcellular structures, and whole cells co-localization, ratiometric imaging, and 3D analysis. In daily a group meeting, students present and discuss their results with the instructors, thereby developing troubleshooting skills and expertise in assessing the quality of quantitative measurements. Students use state-of-the-art microscope systems, including point-scanning and spinning disk confocal, TIRF, multi-photon, light sheet, and super-resolution microscopy. The course instructors are internationally recognized leaders in microscopy, and invited seminar speakers provide examples of the use of quantitative microscopy techniques. Quantitative Imaging: From Cells to Molecules provides training in quantitative microscopy that is crucial for advancing the mission of the NCI.